嗜热盐碱厌氧菌Natranaerobius thermophilus独特的多极端环境适应机理

The poly-extremophile Natranaerobius thermophilus (DSM 18059T)is an unusual bacterium among the extremophiles because it is poly-extremophilicrequiring concomitantly anaerobic, halophilic, alkaliphilic and thermophilic growthconditions, i.e. growing optimally at 3.9M Na+, pH9.5 and 53oC. This bacterium is an excellent model for understanding survival and adaptation strategies under multiple extreme environmental stress. In the combination of multi extremes, they extend the boundary of life conditions. These poly-extremophiles also function as models for early life and for possible extraterrestrial life. The LONG TERM GOAL of this proposal is to understand the novel mechanisms which responsible for the physical and chemical boundaries for life under multiple extreme environmental conditions. The predominant compatible solutes including glycine betaine and glutamate exacted from N.thermophilus have been determined and the completely sequenced genome of N.thermophilus was investigated. On the basis of our preliminary data, three SHORT TERM objectives are proposed to elucidate the molecular mechanisms which contribute to the adaptation in the poly-extremophile N.thermophilus: ① Functional characterization of the osmolyte glycine betaine synthesizing enzymes GSMT and SDMT from N. thermophilus; ② Elucidation of the differential expression of genes （RNA-Seq）and proteins （2D-DIGE）associated with metabolic features to assess the regulatory network in response to variations in the multiple stressors; ③Validation of various groups of the potential genes and proteins using Real time-qPCR and Western blotting related with the unique adaptive mechanism and regulatory network to improve the genome annotation and reconstruct a new model for the global network in N. thermophilus. This study will offer a novel theory of the boundaries for life under multiple extreme environmental conditions. The results of the work will also offer insight into potential biotechnology applications for reactions under combined stress.

嗜热盐碱厌氧菌N.thermophilus最佳生长条件为：盐度3.9M Na+、pH 9.5和53oC。该菌株极为独特的生命存在原理，可以更好地认知生命极限，为揭示生命的起源与演化提供新线索。我们已确定了该菌株细胞内相容性溶质的种类和含量；深入解析了全基因组序列。拟在此基础上：①对被赋予"新功能的甘氨酸甜菜碱合成酶（GSMT和SDMT）"基因进行原核表达及功能鉴定，阐明它们在多极端条件下的分子适应机制；②应用转录组学（RNA-Seq技术）和蛋白组学（2D-DIGE技术）剖析不同极端条件下转录表达谱和蛋白表达谱的差异，与生理代谢表型相关联，探讨它们的调控网络响应模式；③对候选基因群和蛋白群进行功能验证，确定它们在网络调控中的定位和重要功能；最终可完善全基因组注释，构建新的基因网络结构及调控代谢模型。本课题可为生命现象存在的本质提供新颖的理论观点，也可为生物技术的潜在应用提供新基因/蛋白原件。

嗜热盐碱厌氧菌N.thermophilus 最佳生长条件为：盐度3.9M Na+、pH 9.5 和53ºC。该菌独特的生命存在原理，可以更好地认知生命极限。在本项目的资助下，我们确定了①该菌依赖于‘内盐’和‘相容性溶质’策略适应极端条件。其中内盐主要为K+；相容性溶质主要为甘氨酸甜菜碱和天冬氨酸；②在大肠杆菌中克隆表达了被赋予“新功能的甘氨酸甜菜碱合成酶（GSMT和SDMT）”基因，表达的蛋白被纯化及功能被鉴定等，阐明它们在多极端条件下的分子适应机制；③应用蛋白组学新技术（iTRAQ技术）分别测定在以下不同嗜极条件下（Ⅰ. 2.5M，3.1M，3.7M和4.3MNa+；Ⅱ. 3M Na+ pH8.8和42°C和4M Na+ pH9.8和52°C；Ⅲ. 3.3 M Na+或4.5 M Na+且在pH9.5和52ºC下；pH8.5或pH10.5且在3.9MNa+和52ºC下；37ºC或54ºC且在3.9M Na+和pH9.5条件下）深入分析了这些蛋白表达的差异，获得了很多抗盐碱热基因；④同时，利用RNA-seq新技术及ddPCR新技术（数字PCR技术）对蛋白组中的差异蛋白从RNA水平进行了进行了验证。综上，本课题从多个层面解析N.thermophilus独特的生物学特点和极强的多嗜极适应能力，为探索生命现象存在的本质提供新颖的理论观点，也可为生物技术的潜在应用提供新基因/蛋白原件。